Kinetoplastid RNA editing inserts and deletes uridylate (U) residues in mitochondrial pre-mRNAs of trypanosomatids to produce mature mRNA. Small guide RNAs (gRNAs) specify the edited sequence. RNA editing proceeds by a mechanism involving endonucleolytic cleavage of pre-mRNA, addition or removal of Us at the 3' end of the 5' cleavage product, and finally, ligation of the cleavage products. The details of this process are still unclear; it is not certain how sequence information is transferred from gRNA to pre-mRNA. The goal of the proposed research is to elucidate the precise mechanism by which the edited sequence is determined. The primary research tools used will be an in vitro assay for RNA editing developed previously, and a variation, the precleaved editing substrate, in which the pre-mRNA is introduced as two cleavage fragments. To examine the role of guiding nucleotides in insertion editing, gRNAs will be mutated such that they can no longer serve as an RNA template for U addition, to see whether they can still guide U addition. The ability of nucleotides other than U to be added to a 5' cleavage product, in an editing context, will be assessed. Secondly, the contributions of the U-addition/removal and RNA ligation steps in specifying the number of Us inserted or deleted will be measured, using precleaved editing assays under reaction conditions allowing only one step to proceed. Finally, regions of complementarity between gRNA and pre-mRNA will be disrupted or created to determine the importance and characteristics of RNA-RNA duplex structures in editing. A thorough understanding of the mechanism of RNA editing in trypanosomatids may lead to novel therapeutic strategies against these parasites, and measures to stop their propagation by disrupting their cyclic transmission between insect and mammalian hosts.